Open Access Research Mutation screening of the TPO gene in a cohort of 192 Chinese patients with congenital hypothyroidism Chunyun Fu,1,2 Bobo Xie,1,2 Shujie Zhang,1,2 Jin Wang,1,2 Shiyu Luo,1,2 Haiyang Zheng,1,2 Jiasun Su,1,2 Xuyun Hu,1,2 Rongyu Chen,1,2 Xin Fan,1,2 Jingsi Luo,1,2 Xuefan Gu,3 Shaoke Chen1,2 To cite: Fu C, Xie B, Zhang S, et al Mutation screening of the TPO gene in a cohort of 192 Chinese patients with congenital hypothyroidism BMJ Open 2016;6:e010719 doi:10.1136/bmjopen-2015010719 ▸ Prepublication history for this paper is available online To view these files please visit the journal online (http://dx.doi.org/10.1136/ bmjopen-2015-010719) CF and BX contributed equally Received December 2015 Revised 26 March 2016 Accepted April 2016 ABSTRACT Objectives: Defects in the human thyroid peroxidase (TPO) gene are reported to be one of the causes of congenital hypothyroidism (CH) due to dyshormonogenesis The aim of this study was to examine the TPO mutation spectrum and prevalence among patients with CH in the Guangxi Zhuang Autonomous Region of China and to define the relationships between TPO genotypes and clinical phenotypes Methods: Blood samples were collected from 192 patients with CH in the Guangxi Zhuang Autonomous Region, China and genomic DNA was extracted from peripheral blood leucocytes All exons of the 10 common CH-associated genes including TPO together with their exon-intron boundaries were screened by next-generation sequencing (NGS) The effect of the novel TPO mutation was investigated by ‘in silico’ studies Results: NGS analysis of TPO in 192 patients with CH revealed different variations in individuals (2/192, 1%) Sequencing other CH candidate genes in the patients with TPO variants revealed that patient was homozygous for c.2422delT TPO mutation combined with double heterozygous DUOX2 pathogenic variants ( p.R683L/p.L1343F) and patient was triallelic for TPO pathogenic variants ( p.R648Q/p.T561M/p T561M) The present study identified a novel TPO variation c.1682C>T/p.T561M; and four known mutations: c.2422delT/p.C808Afs×24 and c.1943C>T/ p.R648Q in TPO, c.2048G>T/p.R683L and c.4027C>T/ p.L1343F in DUOX2 Conclusions: Our study indicated that the prevalence of TPO mutations was 1% among studied Chinese patients with CH More than two variations in one or more CH-associated genes can be found in a single patient, and may, in combination, affect the phenotype of the individual A novel TPO variation c.1682C>T/p T561M was found, thereby expanding the mutational spectrum of the gene For numbered affiliations see end of article Correspondence to Dr Shaoke Chen; chenshaoke123@163.com INTRODUCTION Congenital hypothyroidism (CH) is a common endocrine disorder with prevalence Strengths and limitations of this study ▪ We conducted one of the largest TPO mutation screenings, in a cohort of 192 patients with congenital hypothyroidism (CH), in the Guangxi Zhuang Autonomous Region, China ▪ We identified that the prevalence of TPO pathogenic variants was 1% among patients with CH ▪ More than two variations in one or more CH-associated gene can be found in a single patient, and the coexistence of multiple mutations may exacerbate the severity of the hypothyroid condition ▪ We found a novel TPO variation, thereby expanding the mutational spectrum of the gene ▪ The relationships between TPO genotypes and clinical phenotypes could not be clearly determined because only two patients were identified to be mutation-positive participants and both had multiple variations ranging from 1:2000 to 1:4000 newborns.1 Apart from iodine deficiency, which is still a major cause for endemic hypothyroidism in neonates,2 the sporadic CH cases can be classified into two groups: (1) disorders of thyroid gland development (thyroid dysgenesis) that have been linked to mutations in TSHR, PAX8, NKX2.1 and FOXE1 genes,3 which account for the majority of cases (80– 85%)3 4; and (2) abnormalities in thyroid hormone synthesis (dyshormonogenesis), which account for the remaining 15–20% of cases,5 and are associated with mutations in DUOX2, TG, TPO, SLC5A5, SLC26A4 and IYD genes.6 Defects in the human TPO (NM_000547) gene are reported to comprise some of the most common causes of thyroid dyshormonogenesis (TDH).7–10 TPO, a thyroid-specific haeme peroxidase localised in the apical membrane of thyrocytes, plays a vital role in thyroid hormone biosynthesis CH caused by mutations in the TPO is an autosomal Fu C, et al BMJ Open 2016;6:e010719 doi:10.1136/bmjopen-2015-010719 Open Access recessive disorder, and most patients with biallelic TPO mutations have permanent congenital hypothyroidism (PCH) Up to now, about 100 mutations have been reported and recorded in the HGMD (http://www hgmd.cf.ac.uk/ac/index.php), but the genotype–phenotype relationship has not yet been fully established, and little is known about its mutational spectrum and prevalence among Chinese patients with CH We performed the TPO gene screening in a cohort of 192 patients with CH in Guangxi Zhuang Autonomous Region, China Illumina Amplicon Viewer V.1.3 and MiSeq Reporter V.2.3 software were used for data analysis, and the SnpEff12 was used for variant annotation Polyphen 213 and MutationTaster14 were used to evaluate the pathogenicity of the novel mutations DNAMAN software V.8 was then used to carry out multiple sequence alignment of the TPO family protein from different species In addition, a cohort of 300 ethnicity-matched healthy participants was used to assess the variant frequencies in the normal control All control participants had normal FT4 and TSH levels MATERIALS AND METHODS Patients We enrolled 192 patients (101 females and 91 males) with CH; the previous 45 patients with CH collected for DUOX2 mutation screening were not included in this study.11 Most patients were initially identified by neonate screening among 623 000 newborns in the Guangxi Zhuang Autonomous Region, China, from October 2009 to June 2014 The thyroid gland was normal in size and position in 73 of these patients; 72 patients had increased and 47 showed decreased size of the thyroid gland Newborn screening was performed with filter paper for CH between 72 h and days after birth Blood samples were collected from the heel and the thyroidstimulating hormone (TSH) level was measured by timeresolved fluorescence assay (Perkin Elmer, USA) Newborns with increased TSH (TSH ≥8 mIU/L) levels observed during neonatal screening were followed-up for further evaluation Serum TSH and FT4 were determined by electrochemiluminescence assay (Cobas e601, Roche Diagnostics, USA) Diagnosis of CH was based on elevated TSH levels (TSH >10 mIU/L) and decreased FT4 levels (FT4 40× Also, no deletion or duplication of exons in TPO was detected by manual read depth inspection In this study, we identified three different TPO variants in two individuals Sequencing of other CH candidate genes in the two patients with the TPO mutations showed that patient was homozygous for c.2422delT TPO mutation combined with double heterozygous DUOX2 mutations ( p.R683L/ p.L1343F), and patient was triallelic with TPO pathogenic variants ( p.R648Q/p.T561M/p.T561M) All variants were confirmed by Sanger sequencing (figures Fu C, et al BMJ Open 2016;6:e010719 doi:10.1136/bmjopen-2015-010719 Open Access Figure Pedigree of the affected family and Sanger sequencing for validation of the mutations detected by the next-generation sequencing platforms and 2) The present study identified a novel variation: p T561M in the TPO; and four known mutations: c.2422delT and p.R648Q in TPO, p.R683L and p.L1343F in DUOX2 Polyphen2 and Mutation taster predicted that the novel variation p.T561M would have deleterious effects by damaging the TPO protein Additionally, the variant was not detected in our normal control population DNAMAN software was then used to carry out multiple sequence alignment of the TPO family protein from different species; the identified variation was found to be located in the highly conserved region of TPO (figure 3) These all suggested that the amino acid substitution might be due to a pathological mutation In Silico predictions of functional impact of the T561M on the TPO protein The generated 3D homology models based on 1mhl template (44% sequence similarity) covered 50% of the full sequence of TPO protein Verify3D showed that the model had passed as a good model by having 86.6% of residues with an average 3D to 1D score of more than 0.2 Ramachandran plots from Procheck showed that the model had three amino acid (Gly331, Glu384 and Lys627) residues located in the disallowed regions However, these amino acid residues were distant from the active site Consequently, the homology model was acceptable for subsequent analyses The threonine 561 residue is located within a highly conserved region of TPO, which suggests an important role in the function and/or structure of TPO Comparison between predicted tertiary structures of the wild type and mutant proteins revealed that the substitution, p.T561M, breaks two hydrogen bonds with an asparagine in position 557 and another hydrogen bond with a glutamic acid in position 558 Significant structural alterations in the annotation were also detected in the p.Thr561Met mutant (figure 4) As a result, these changes will disturb the secondary structure and affect the function of TPO protein Clinical features and laboratory test results of the patients The clinical features and laboratory results are summarised in table Patient was born at full-term to non-consanguineous parents His birth weight was 3000 g and length 47 cm There was no family history of thyroid disease Newborn screening at the age of days Figure Pedigree of the affected family and Sanger sequencing for validation of the mutations detected by the next-generation sequencing platforms NA, data are not available Fu C, et al BMJ Open 2016;6:e010719 doi:10.1136/bmjopen-2015-010719 Open Access Figure Multiple sequence alignment of TPO from different species The arrow indicates that the threonine 561 residue is located within a highly conserved region revealed a high level of TSH (78.6 mIU/L) He was therefore re-evaluated at hospital at the age of 13 days A serum TSH level of >100 mlU/L (normal range 0.7– 10 mIU/L), FT4 level of 1.92 pmol/L (normal range 12–32 pmol/L) and FT3 level of 1.68 pmol/L (normal range 2.65–9.68 pmol/L) confirmed the diagnosis Ultrasound examination showed an enlarged thyroid gland (right lobe 3.4×1.5×1.4 cm; left lobe 3.2×1.4×1.3 cm) L-T4 replacement therapy was started immediately at an initial daily dose of 12 μg/kg and adjusted according to the serum TSH, FT4 and FT3 levels The diagnosis of PCH was confirmed due to a high TSH level (38.87 mIU/L) after temporary withdrawal of L-T4 therapy for weeks at the age of 2.3 years The L-T4 replacement therapy was then resumed The patient is now 3.1 years of age and receives a daily dose of μg/kg L-T4 His physical and intellectual development is appropriate to his age Family studies showed that his father carried a heterozygous TPO mutation c.2242delT and a heterozygous DUOX2 mutation p R683L but with no thyroid phenotype, and his mother harboured the heterozygous TPO mutation c.2242delT without abnormal thyroid phenotypes (figure 1) Patient was diagnosed with CH in our outpatient paediatric endocrinology clinic The boy was 4.5 years old at the time; he was 98 cm (